Cellular Intoxication Pathway of Cytolethal Distending Toxin

细胞致死膨胀毒素的细胞中毒途径

• 批准号: 8322033
• 负责人: Kenneth Alan Bradley
• 金额: $ 43.18万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8322033
• 关键词: Address; Area; Bacterial Proteins; Bacterial Toxins; Binding; Biology; California; Carrier Proteins; Cell Cycle; Cell Cycle Arrest; Cell Line; Cell Nucleus; Cell Surface Receptors; Cell membrane; Cell physiology; Cell surface; Cells; Cellular biology; Comparative Study; Complement; Complex; Cytokinesis; Cytosol; DNA Damage; DNA Repair; Data; Deoxyribonuclease I; Destinations; Endocytosis Pathway; Event; Family; Foundations; Future; Genes; Genetic; Genetic Screening; Gram-Negative Bacteria; Hemophilus ducreyi; Illinois; Immune Sera; Integration Host Factors; Intoxication; Intracellular Transport; Knowledge; Los Angeles; Mammalian Cell; Membrane Protein Traffic; Methodology; Modeling; Molecular; Mutagenesis; Nature; Operon; Organelles; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Process; Production; Property; Protein Subunits; Proteins; Reagent; Receptor Cell; Research; Research Personnel; Resolution; Role; Route; Signal Transduction; Structure; System; Time; Toxin; Translating; Universities; Vesicle; Vesicle Transport Pathway; Work; ataxia telangiectasia mutated protein; base; cytolethal distending toxin; design; extracellular; holotoxins; human H2AX protein; human disease; insight; member; molecular carrier; mutant; novel; novel therapeutics; pathogen; pathogenic bacteria; receptor; receptor binding; research study; tool; trafficking; uptake

DESCRIPTION (provided by applicant): The intracellular-acting bacterial protein toxins have evolved various means to interact with and enter mammalian cells, taking advantage of existing host cellular processes. Studies of the mechanisms by which bacterial toxins are internalized have provided fundamental advances in several areas of cell biology, including receptor biology, mechanisms and pathways of endocytosis, vesicle trafficking, and membrane translocation. Here, we propose studies focused on one member of an emerging family of toxins called the cytolethal distending toxins (CDTs), which are unusual in their intracellular localization, modulatory activities, and overall structures. CDTs are multi-subunit toxins that are generated by a diverse group of pathogenic Gram-negative bacteria and function by modulating the host cell cycle. Active CDT holotoxins are heterotrimeric complexes of three protein subunits, generally encoded by three contiguous genes (cdtA, cdtB, cdtC) in a single operon. To exert their cyclomodulatory effects on cells, CDTs must be taken up from the cell surface and transported intracellularly in a manner that ultimately results in localization of the enzymatic subunit CdtB to the nucleus. However, the molecular details and mechanism by which CDTs exploit existing uptake and transport pathways to gain access to the cytosol, and ultimately the nucleus, are poorly understood. This application addresses these current gaps in knowledge and represents a collaborative proposal between investigators at the University of California at Los Angeles (Dr. Bradley) and the University of Illinois (Dr. Blanke) to investigate the molecular basis of CDT cellular intoxication. Two Aims are proposed that address hypotheses regarding the cell surface binding (Aim 1) and intracellular transport (Aim 2) of CDTs required for cellular intoxication. Aim 1 investigates the molecular determinants of the initial CDT-cell binding contributed by both the host (Aim 1.1) and toxin (Aim 1.2). Aim 2 focuses squarely on the host cellular requirements for uptake and trafficking of CDT, utilizing hypothesis driven (Aim 2.1) and forward genetic (Aim 2.2) approaches. Importantly, the heterotrimeric nature of CDTs, as well as the localization of the catalytic CdtB subunit to the nucleus, are unique features of the CDTs. Therefore, identification of host cell determinants and trafficking pathways that are important for CDT intoxication is predicted to reveal novel host cell requirements and/or routes for transporting proteins from the cell surface to the nucleus and provide insight into toxin-host interactions.

描述（由申请方提供）：细胞内作用的细菌蛋白毒素已经进化出各种方式，利用现有的宿主细胞过程与哺乳动物细胞相互作用并进入哺乳动物细胞。对细菌毒素内化机制的研究已经在细胞生物学的几个领域提供了根本性的进展，包括受体生物学、内吞作用的机制和途径、囊泡运输和膜易位。在这里，我们提出的研究集中在一个新兴的家庭的毒素称为致死性膨胀毒素（CDTs），这是不寻常的细胞内定位，调节活动，和整体结构。CDTs是由多种致病性革兰氏阴性菌产生的多亚基毒素，通过调节宿主细胞周期发挥作用。活性CDT全毒素是三个蛋白质亚基的异源三聚体复合物，通常由单个操纵子中的三个连续基因（cdtA、cdtB、cdtC）编码。为了发挥其对细胞的周期调节作用，CDTs必须从细胞表面摄取并以最终导致酶亚基CdtB定位于细胞核的方式在细胞内转运。然而，CDTs利用现有的摄取和转运途径进入细胞质并最终进入细胞核的分子细节和机制知之甚少。本申请解决了这些知识的差距，并代表了加州大学洛杉矶分校（布拉德利博士）和伊利诺伊大学（布兰克博士）的研究人员之间的合作建议，以调查CDT细胞中毒的分子基础。提出了两个目标，解决有关细胞表面结合（目标1）和细胞内转运（目标2）的细胞中毒所需的CDTs的假设。目的1研究由宿主（目的1.1）和毒素（目的1.2）贡献的初始CDT细胞结合的分子决定因素。目标2直接关注宿主细胞对CDT摄取和运输的要求，利用假设驱动（目标2.1）和正向遗传（目标2.2）方法。重要的是，CDT的异源三聚体性质以及催化CdtB亚基定位于细胞核是CDT的独特特征。因此，鉴定对CDT中毒很重要的宿主细胞决定簇和运输途径预计将揭示新的宿主细胞要求和/或将蛋白质从细胞表面转运到细胞核的途径，并提供对毒素-宿主相互作用的了解。